Recent work from our group and others implicate aberrant neutrophil extracellular trap (NET) formation, the programmed release of chromatin fibers in complex with antimicrobial peptides, as a potentially crucial phenomenon involved in the induction of autoimmune responses, organ damage and premature cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Our data are supported by recent studies also linking NETosis to idiopathic atherosclerosis and to venous thrombosis. This competing renewal application will determine the in vivo importance of aberrant NETosis in CV damage and other manifestations of SLE, with obvious implications for medical therapy in patients affected by this condition. Aim 1: Explore the impact of NET inhibition on murine models of SLE vascular damage. While molecular events implicated in NET formation are incompletely understood, recent evidence indicates that histone citrullination is crucial for chromatin decondensation leading to NETosis6. Neutrophils express high levels of peptidylarginne deiminase-4 (PAD4), the enzyme that catalyzes histone citrullination7. Mice that lack PAD4 have decreased NETosis upon stimulation8. These observations indicate that PAD4 is crucial for NETosis and could be explored as a target in SLE and its associated vascular complications. Our collaborator, Dr. Paul Thompson (Scripps), has developed a novel PAD inhibitor N-?-benzoyl-N5-(2-chloro-1-iminoethyl-L-ornithine amide (Cl-amidine) that inhibits nuclear PAD4 with low ?M potency9,10. Cl-amidine inhibits NET formation in human and murine SLE neutrophils (see preliminary data) and has previously been well-tolerated in other murine inflammatory models. We will utilize chemical PAD4 inhibition and available PAD4 knockout (KO) models to block NETosis in murine SLE and in models of lupus with accelerated atherogenesis, allowing us, to assess the in vivo impact of NET inhibition on models of lupus-associated vascular damage: carotid thrombosis, neoangiogenesis, endothelial-dependent vasorelaxation and atherosclerosis. We predict that blocking NET formation will significantly reduce vascular damage in SLE. Aim 2: Determine if other SLE manifestations can be abrogated by NET inhibition. Recent data from various groups supports the concept that NETosis is a stimulus for IFN-? production, a phenomenon that may promote autoimmunity3,13,14; further, there are emerging hints that IFN-? may stimulate NETosis, thereby resulting in a harmful, self-perpetuating cycle. As NETs can damage the vasculature directly, Aim 1 is a logical place to begin our assessment. However, we anticipate that NET inhibition will also have a global impact on SLE disease activity and on type I IFN responses. We will assess the role of aberrant NETosis in autoantibody (autoAb) synthesis, induction of type I IFN-responses, adaptive immune responses and organ inflammation and damage in murine SLE, with a particular focus on kidney and skin damage. Aim 3: Determine the association of aberrant neutrophils, NETs and anti-neutrophil Abs in surrogate markers of endothelial dysfunction and CV risk in SLE patients. The association of neutrophil:platelet aggregates (NPAs), low-density granulocytes (LDGs), NETosis and autoAbs to neutrophil-derived immunostimulatory products, with endothelial dysfunction, subclinical atherosclerosis (carotid and coronary), type I IFN signatures and surrogate markers of vascular damage will be determined in cross-sectional and longitudinal analyses in a well-characterized CV Lupus Cohort established at the U. of Michigan. Results from this proposal will provide crucial information regarding the role of neutrophils and aberrant NETosis in the pathogenesis of autoimmune responses and organ damage in SLE, and may lead to changes in the therapeutic armamentarium in individuals with this devastating condition. If we confirm our hypothesis and extend the knowledge regarding mechanisms of effect in the studies proposed herein, Cl-amidine or other PAD-4 inhibitors may represent the first of a novel class of disease-modifying drugs for lupus that target a recently described process which may be crucial in autoimmune responses and organ damage in SLE.